Pregnatetraenes and esters thereof



PREGNATETRAENES AND ESTERS THEREOF David H. Gould, Leonie, Emanuel B. Hershberg, West Orange, and Elliot Shapiro, lrvington, N. J., assignors to Schering Corporation, Bloomfield, N. J., a corporation of New Jersey No Drawing. Continuation of application Serial No. 581,265, April 30, 1956. This application November 25, 1957, Serial No. 698,337

18 Claims. (Cl. 2613-39145) atent O The compounds of our invention are represented by unsaturated pregnanes having the following general formula:

wherein X is a member of the group consisting of (l-LoiOR), (H,BOR), and O; R is a member of the group consisting of H and acyl; Z denotes the arrangement of C8, C-9, and C14 consisting of r CB\/Cl4 (MK/C14 Ca Cs and W is a member of the group consisting of H, F, Cl

and Br.

The compounds of the'general formula which are devoid of a halogen group at C-9 exhibit an interesting distribution of pharmacological properties. In general, those compounds possessing an OH group at C 17 together with either a ,eOH or keto function at 0-11 are adrenocorticoid in their action. The compounds are useful in the treatment of inflammation, arthritic diseases and certain other collagen diseases such as disseminated lupus, polyarteritis and the like, and are advantageously employed over known corticoids, such as cortisone and desoxycorticosterone in that they arerelatively devoidof electrolyte retention. Thus, following administration of these pregnatetraenes there is relatively littlesodium retention and concomitant edema, which is so common with many of the adrenocortical hormones and corticoids heretofore in use. In combination with vmineralocorticoids such as desoxycorticosterone, our compounds proice vide and enhance resistance to stress and aid in-the control of carbohydrate metabolism. Corollary to the foregoing, these pregnatetraenes are useful in suppression of pituitary ACTH output.

A further important application of these new steroids is in the treatment of'allergicphenomenasuch as asthma, and certain types of perennial rhinitis, angioneurotic edema, and drug sensitivities, which do not respond to usual antihistaminic therapy. Moderate doses of these substances are effective in giving prompt relief to an acute asthmatic attack, or resistant status asthmaticus, which might otherwiseprove fatal.

The 9 t-halogenated compounds of the generalformula are useful in maintaining bilateral adrenalectomized patients and Addisonians.

Topical preparations of the compounds of-the invention in the form of ointments, suspensions and the like, lend themselves to ophthalmologicaluse where they are used toblock inflammatory reactions arising from allergic disorders, as well as bacterial or toxic-origins (such as acute iritis). p

Those compounds of the general formula-possessing an acyloxy group at C-17, especially in combination with a Cll ester function and/or a C- Z1 ester group such as 1,4,6,8(9) pregnatetraene llfi,17a,21 triol 3,20- dione-ll-formate-(or acetate)-17"-acetate and the corre sponding C-21 esters, are progestational agents showing 0.5-1.5 times the activity of progesterone.

Our new substances may be administered for their particular therapeutic use either orally or parenterallyin the form of tablets, capsules, aqueous or oil suspensions or solutions. In the usual cortic'oid treatment suclras antiinflammatory therapy, replacement therapy and the like, the daily dose is 5-10 mg. depending upon the extent of therapy required. In the treatment of allergic phenomena, a higher daily dose in the range'of 10-20 mg. is required to effect relief from acute bronchial asthma.

We have found that esterification of thecompounds of our invention in the C-21 position allows for an extension of the-duration of activity over that exhibited by the free C-Zl alcohol. For example, lo'wer alkanoic acid radicals and cycloaliphatic alkanoic acid radicals such as acetate, propionate, isovalerate, enanthate and cyclopentylpropionate are particularly effective in this instance. Furthermore, certain aryloxy alkanoic ac'id radicals and certain heterocyclic carboxylic acid radicals, when attached in the form of an ester link at'C2-1, providefor a longer duration of activity than is obtained with the'lower alkanoic acid radicals, and thus permit the utilization of a smaller dose to achieve a desired result. Byway of example, such radicals include phenoxyacetate, s'ubstit'tited phenoxyacetates such as 4-bromo-, 4-methyl-, 4-tertiary butyl-, 2,4,5-tn'chloroand 4-methoxy-phenoxyacetates. As examples of the heterocycliccarboxylic acid radicals, We have found that the furoates and substituted furoates including the 5-bromo-, 5-methyl-, and S-t'ert'iary-butyI- turoate radicals are especially valuable.

Since the unesterified compounds ofthe formula togethcr with their monobasic acid esters are essentially insoluble in water, we can overcome this difiiculty and can provide for water solubility by forming non-toxic metal salts such as the sodium salts of monoesters of polycarboxylic acids such as succin'ic, phthalic, tricarballylic, itaconic acids, etc., preferably at-C-21.

The 1,4,6,8(9)-pregnatetraenes of our invention are preferably prepared from 1,4,6-pre'gnatriene-17a,21-diol- 3,20-diones which possess an oxygen function at C-ll. The 1,'4,6-pr'egnatrienes used as starting materials are synthesized'according to the procedure described in copending application of Gould and Herzog, Serial No. 513,901, filed June 7, 1955. After substitution of an anionic radical such as hydroxyl, halide, sulfate, sulfonate, nitrate, etc., for an hydrogen atom on either -8 scribed in the copending application of Hershberg and Herzog, Serial No. 486,028, filed February 3, 1955, gives rise to the corresponding C-S-(or 9) hydroxy derivative. The exact position at which hydroxylation occurs is unknown. However, for the purposes of this process, it is unimportant, since the intermediary compound so obtained from microbiological procedure is dehydrated so as to introduce an additional double bond between (3-8 and C-9. The dehydration is easily accomplished chemically by a variety of agents such as alumina, inorganic bases (sodium hydroxide, sodium bicarbonate, etc.), aqueous acetic acid, p-toluenesulfonic acid in chloroform or by heating in xylene. This sequence of reactions is shown in the following schematic diagram. (The uncertainty of the point of attachment of the hydroxyl group introduced by the microorganism is indicated by the use of a bracket indicating that attachment may be either 0-8 or 0-9.)

CHzOH 0:0 l---on CHzOH heat in xylene) We prefer, however, to use a halogen such as chlorine,

bromine, or iodine as the anionic substituent at C-9 and we also prefer the chemical approach to the overall synthesis. This preferred process is shown in the following equation:

The starting compound V in this instance indicated as a 9a-bromo substance may also be a 9OL-Chl0f0 or fluoroanalog, and is prepared according to the analogous procedure described in the copending application of Gould and Herzog, Serial No. 513,901, filed June 7, 1955. The elimination of hydrohalic acid between C-8 and C-9 is effected in a known manner for dehydrohalogenation (such as by heating the 9tx-halotriene in the presence of acetic acid) gives rise to the 1,4,6,8(9)-pregnatetraene 11fi,l7a,2l triol-3,20-dione 21-tcetate (lb). Saponification of the Zl-ester is easily accomplished by treatment with dilute alkali as indicated, yielding 10. Alternatively, the dehydrohalogenation may be etiected with other reagents such as collidine or sodium acetate in acetic acid on the corresponding ll-keto-analogs of V.

We have also found that the compounds described in copending application, Serial No. 513,901, filed June 7, 1955, are useful intermediates in the preparation of the pregnatetraenes of this invention, in that they lend themselves to the insertion of a halogen atom at the C8 position as shown in the following equation:

In the foregoing a 21-ester such as the acetate of 1,4,6- pregnatriene-17a,21-diol-3,11,20-trione is reacted with a source of positive halogen ion such as bromine or N- bromosuccinimide whereby a bromine atom is introduced at C-8 (VII). Dehydrohalogenation with sodium acetate in acetic acid, for example, gives rise to 1,4,6,8(9)- pregnatetraene-l7a,21-diol-3,11,20-trione 2l-acetate.

If, in this sequence of reactions, the starting material is the llfl-hydroxy analog of V], we prefer to first formylate the llfi-hydroxyl group with formic acid, according to the procedure described by Oliveto et aL, in the copending application, Serial No. 421,948, filed April 8, 1954. After dehydrohalogenation, one obtains the corresponding 1,4,6,8(9)-pregnatetraene containing an llfi-formyloxy group. Saponification is easily eltected by means of alkali or microbiologically, using Flavobacterium dehydrogenans var. hydrolyticum as described in the copending application of Charney, Serial No. 458,661, filed September 27, 1954, now abandoned.

We have found that when the anionic substituent is adjacent to an activated hydrogen atom, it is easily removed with the said hydrogen atom by relatively mild treatment with acid or base which may be accompanied by heating. For example, reagents such as acetic or formic acids, sodium acetate, alkali bicarbonates, carbonates or heating in inert solvents are useful in effecting this dehydrohalogenation. On the other hand, when the substituent is not adjacent to an activated hydrogen atom such as would be in the situation when the starting material is l,4,6-pregnatriene-l1B,l7a,2l-triol-3,20-dione-llformate-Zl-acetate, somewhat stronger measures to effect dehydrohalogenation are necessary. Methods for effecting this type of dehydrohalogenation are exemplified by dehydration with a strong acid; moderate pyrolytic procedures in the presence of organic bases such as collidine, .quinoline, etc., at 150250; t eatme'nt With strong dehyd'roha'loge'nators such as silver or mercury salts in org'anic solvents such as pyridine, collidine, acetonitrile, etc.

In order to prepare the 1,4,6,8(14)--pregnatetraenes of the general formula, we prefer to hydroxylate a 1,4,6- pregnatriene at the (3-14 position, isolate the 14oz-hydroxy compound and dehydrate to the tetraene. Thef'ollowin'g sequence of reactions illustrates a method for preparing the l4oc hydroxy intermediates:

In the above reaction a :Pregna'triehe (VIII).-is subjected to the oxygenating activity ofa microorganism which hydroxylates in the l lac-position such as Curvularia lunata. The product soforrned-is the corresponding 14ozhydroxypregnatriene; 1X, which may be esterified at 'C-21 with acetyl chloride in pyridine, for example.

Alternatively, a 4,6-pregnadiene such as 4,6=pregnidiene- 17oc,2 l-diol-3,11,20-trione may be hydroxylated with Curvu laria .lunata so as to produce the corresponding 14a-hydroxy-pregnadiene. Subjecting the latter compound to the dehydrogenating action of a-dehydrogenating organism, such as C. simplex or B. sphaericus affords 1,4,6-pregnatriene-14u,17a,21-triol-3,l1,20-trione. Conversion of the 14a-hydroxy compounds to 1,4,6,8(14)- pregnatetraenes is carried out in a facile manner. In general, the major step -is the removal of an anionic substituent at'C-14 together with the hydrogen atom at C-8 which is activated by the conjugated unsaturation in rings A and B, leading to the insertion of a double bond between C8 and (1-14 which is in conjugation with the previous unsaturation. We generally prefer as the anionic substituent a 14ix-hydroxylgroup.

(limo H C=O OH Pyridine o:

,Dehydrate acid in ice, and extracted with methylene chloride.

As dehydration procedures there are used the following': :saponificat-ion or -treatmentwith mild baseyt'reatby heating-in toluene, xylene, collidine and the like. It

is to be noted that stronger treatment with acid orbase in the case of 1,4,6,8(14)-pregnatetraene-17ot,2l-diol- 3,-11,20.-trione isomerizes the terminal double bond to give 1,4,6,8(9) pr'egn'atetraene 17a,21-diol-3,11 ,20-trione.

Partially esterifiedpregnatetraenes may be prepared according-to a variety of prescribedprocedures. We preferingeneral to fully es'terify all hydroxyl groups which are present and then selectively saponify as required. For example, 1,4,6,8(9)-pregnatetraene--11,8,17a-21-triol- 3,20-dione may be acylated with reagents such as acetic anhydride and acetic acid whereby ac'etyl'a't'ion occurs/at the -11,-17 and 2-l-position aifordingthe corresponding triacetate. subjecting the triacetate so obtained to mild hydrolytic conditions results in 'the formation of the corresponding 1 1 3-'17oi-,21-t'riol-1I,l7diacetate. Instead of the aforementioned acylatin'g ag'e'nt, other agents such as formic acid in the presence of p-toluenesulfonic acid are applicable and the corresponding form'a'te esters are obtained.

An llx-keto 17,21 diester may be reduced either chemically or microbiologically according to known methods whereby the. corresponding 11fi-hydroxy-17,21 diester is produced. Other methods 'of preparing various combina- 'tions are set forthin the following "examples.

The fc'illowin'g examples are presented toillustrate various methods for preparing the compounds'of ourinvent-ion. It will be appare'nt-to'a chemist skilled in the art thatyarious modificationsare inessence "no more than "equivalents. Thus our inventionis limited only as defined in the appending claims.

EXAMPLE 1 1 ,4 ,6 ,8(' 9) -'pregnatetraene-1 7 oc,21 -diol'-3 ,1 1 ,20-tri0ne I. 21 acetate (A) A mixture of 500 mg. of 9a-chloro-1,4,6-pregnatriene-l7e,2 l-diol-3,11,20-trione Zl-acetate (prepared by substituting hydrogen .chloride for hydrogen fluoride in the procedure described in the copending application of Gould-and Herzog, Serial No. 513,901, filed June 7, 1955-) and 10ml. of 'y-COllOdine is refluxed for 15 minutes. The cooled mixture'is poured into excess sulfuric The organic layer is washed with water until neutral, dried and concentrated to a small volume. The concentrate is chrcirnatographed on activated magnesium silicate and fixed by 'using'hexane as thedeveloper. Chromatographic elution with ether-hexane mixtures affords the compound of this example in the fraction where ether isthe eluting solvent. Purification is effected by recrystallization from acetone-hexane.

(B) Alternatively, the compound of this example may be prepared by treating a solution of 300 mg. of 9abromo 1,4,6-pregnatriene-17a,2l-diol-3,l1,20-trione 21- acetate in 5 ml. of chloroform and 10 ml. of acetic acid with 56 mg. of anhydrous sodium acetate in 5 ml. of acetic acid. The mixture is refluxed for two hours, .concentrated in vacuo and poured into water. The resultant precipitate from methylene chloride-hexane affords the tetraene of this example.

(C) A further procedure for the preparation for the compound of this example is as follows: To a stirred solution of 1.0 g. of 1,4,6-pregnatriene-17a,21-diol-3,11, ZO-trione ZI-acetate in 10 ml. of methylene chloride is dropwise added 0.22 g. of bromine in 5 ml. of acetic acidat a rate governed by the disappearance of color 'due to excess bromine. After an additional 15 minutes s'tir-.

7 ring, the solution is concentrated in vacuo to remove methylene chloride and the residual mixture is poured into water. The solid is removed by filtration, washed with water, dried, and crystallized from acetone-hexane to give the intermediate 8- (or 9-) bromo-l,4,6-pregnatriene-17a,2l-diol-3,11,20-trione Zl-acetate.

The intermediary 8-bromo-1,4,6-pregnatriene-17a,2ldiol-3,11,20trione 2l-acetate may alternatively be prepared according to the following:

A sample of 1 g. of 1,4,6-pregnatriene-l7a,21-diol- 3,11,20-trione 2l-acetate is dissolved in 50 ml. of a 1:1 mixture of carbon tetrachloride and chlorobenzene. The solution is brought to a boil by irradiation with a 300 w. photofiood lamp and treated with 0.40 g. of N-bromosuccinimide. The mixture is boiled with irradiation for 10-15 minutes until the solution no longer gives a starch iodide test. The mixture is cooled, filtered, washed with water and evaporated to dryness in vacuo. Crystallization from methylene chloride-hexane affords the 8- (or 9-) bromc-1,4,6-pregnatriene-17u,2l-diol-3,11,20-trione 2l-acetate. The 8-bromo-pregnatriene intermediate so obtained is dehydrohalogenated with sodium acetate in acetic acid according to the procedure described in part B yielding 1,4,6,8(9)-pregnatetraene-17a,21-diol-3,11,20- trione 2l-acetate.

EXAMPLE 2 1 ,4,6,8 (9 -pregnatetraene-1 7u,21-a'i0l3,11,ZO-trione EXAMPLE 3 1,4,6,8 (9)-pregnatetraene-11fi,170:,21-

triol-3,20-dine 21 -acetate (A) A sample of 0.2 g. of 9a-bromo-l,4,6-pregnatriene-ll,5,l7a,2l-triol-3,20-dione 21-acetate is dissolved in ml. of acetic acid containing 0.4 g. of sodium acetate and the solution is boiled 30 minutes. The solution is cooled and poured into water and the separated solid is extracted with methylene chloride. The organic layer is washed with aqueous sodium bicarbonate and water F till neutral, and dried with anhydrous magnesium sulfate. The dried solution is filtered, concentrated and chromatographed on activated magnesium silicate. The fraction eluted with 10-25% methylene chloride in ether is crystallized from ethyl acetate to give l,4,6,8(9)-pregnatetraene-l 1,8, 170:,21-t1i01-3,20-di0116 2l-acetate.

(B) Alternatively, the compound of this example is prepared in the following manner. One gram of 1,4,6- pregnatrienc-l1B,l7a,2l-triol-3,20-dione ll-formate 21- acetate is brominated with 0.21 g. of bromine and the product is crystallized from aqueous acetone to give 8- bromo 1,4,6 pregnatriene 1l;8,17or,2l triol 3,20- dione ll formate 2l-acetate according to the procedure described in Example lC. A sample of 0.8 g. of the bromopregnatriene so obtained is dissolved in 'ml. of -collidine and treated with 1.5 g. of silver nitrate. The solution is warmed on the steam bath at 90-95 for 6 hours and poured into ice water. The mixture is acidified with dilute sulfuric acid and extracted with methylene chloride. The combined methylene chloride washes are washed neutral with water, dried over magnesium sulfate, filtered and evaporated. The residue is crystallized several times from acetonehexane to give l,4,6,8(9)-pregnatetraene-115,17a,2l-trio1- 3,20-dione ll-formate 21'acetate.

8 EXAMPLE 4 1,4,6,8 (9) -pregnatetraene-l 113,] 741,21 -tri0l-3,20-di0ne A sample of 0.05 g. of the product of Example 3 is saponified and crystallized as in Example 2 to give 1,4,6, 8(9)-pregnatetraene-11/3,l7a,2l-triol-3,20-dione.

Alternatively, the following process is applicable:

A mixture is prepared of 1 g. of yeast extract concentrate and 1 ml. each of 2 M potassium dihydrogen phosphate and 2 M disodium phosphate in each ml. Ten Erlenmeyer flasks (300 ml.) containing 100 ml. each are sterilized and inoculated with flavobacterium dehydrogenans var. hydrolyticum according to the procedure described in copending application of William Charney, Serial No. 458,661, filed September 27, 1954, now abandoned. The flasks are shaken at 30 for 16 hours, and to each is added a solution of 50 mg. of 1,4,6-pregnatriene-llfl,l7or,2l-triol-3,20-dione ll-formate 21-acetate in 5 ml. of methanol. The cultures are shaken at 30 for 24 hours and the combined broths are extracted three times with 300 ml. of methylene chloride and the extract is dried, filtered and evaporated to dryness. The residue is crystallized from acetone to give 1,4,6,8(9)- pregnatetraene-l 1,8,l7a,2l-triol-3,20-dione.

EXAMPLE 5 1,4,6,8 (9) -pregnatetraene-11fl,1 7a,21tri0l-3,20-di0ne triacerale A sample of 500 mg. of the triol of Example 4 is dissolved in a solution of 5 ml. of acetic acid and 5 ml. of acetic anhydride and treated with 50 mg. of p-toluenesulfonic acid. The mixture is allowed to stand overnight and upon dilution with water, the triacetate precipitates. The ester is separated by filtration and recrystallized from ether-hexane solvent, afiording 1,4,6,8(9)-pregnatetraenellfi,17a,21-trio1-3,20-dione triacetate.

EXAMPLE 6 1,4,6,8(9) -pregnatetraene-17a,21-di0l-3,11,20-di0ne diacetate A mixture of 500 mg. of the compound of Example 2 in 5 ml. of acetic anhydride and 5 ml. of acetic acid and 50 mg. of p-toluenesulfonic acid is processed according to the procedure described in Example 5. Recrystallization from ether-hexane mixtures affords the diacetate of this example.

EXAMPLE 7 1,4,6,8(9)-pregnatetraene-11;8,1 7a,21 -tr[0l-3,20-dione triformate To a mixture of 10 ml. of formic acid and 1 g. of the triol of Example 4 is added 100 mg. of p-toluenesulfonic acid. The mixture is chilled in a refrigerator for 48 hours after which time it is diluted with water and the resultant precipitate removed by filtration. The crude triformate so obtained is purified by recrystallization from etherhexane.

EXAMPLE 8 1,4,6,8 (9 -pregnatetraene-I 1 3,1 712,21-0'1'01-320410110 11,] 7-cliformate A solution of 1 g. of the triformate obtained in Example 7 in 20 ml. of absolute ethanol is treated with 100 mg. of p-toluenesulfonic acid monohydrate and the resultant mixture is stirred at 35 to 40 for about 2 hours. The

mixture containing the partially saponified steroid is chilled overnight, diluted with water, and the resultant precipitate removed by filtration. Recrystallization from ether-hexane aflfords l,4,6,8 (9 -pregnatetraenel 1 [9, 1 7a,- 21 triol-3,20-dione-l1,l7 diformate.

EXAMPLE9 1,4,6-pregnatriene-14a,17a,2]-di0l-3,11,20-tri0ne and I,4,6-pregnatriene-8fi,17a,21-triol-3,I1,20-tri0ne One hundred grams of edamine enzymatic digest of lactalbumin, 15 g. of corn steep liquor and 250 g. of

.cer'elose is diluted to l. with tap water and adjusted with No: 8992. The flasks are incubated 24 hours at 28 While shaking on a shake table. To each flask is added a solution of 50 mg. of 1,4,6-pregnatriene-17a,2l-diol-3,11,20- trione (prepared according, tothe procedure described in eopendirig applieationo'f Gould and Herzog, Serial No. 513,901, filed June 7, 1955) dissolved in a minimum amount of acetone. The flasks are again shaken and ineubated at 28 for 24 hours, after which50 ml. of methylene chloride is added to eachfla'sk. After shaking, the organic layer is separated and combined with a second methylene chloride Wash. The combined solutions are dried, filtered and evaporated to a crystalline residue. This residue is triturated with hexane and the hexane withdrawn. Theremainder is dissolved in a minimum amount of methylene chloride (30-50 ml.), and chromatograph'ed on 50 g. of activated magnesium silicate. The column is washed with 500 ml. of anhydrous ether, and

. 1 of methylene chloride followed by five 200ml. fractions of methylene chloride containing 0.5% methanol EXAMPLE 1O 1 ,4,6-pregnatriene-14a,1 70:,21-tri0l-3,11,20-tri0ne 21 -pr0pi0nate A sample of 0.1 g. of the 14a-hydroxy product of Example 9 is dissolved in 5 mloofdry pyridine and chilled to 5. This solution is treated with 50 mg. of propionyl chloride with stirring at 0 for 15 minutes and at 15 for 15 minutes. The mixture is poured into 50 ml. of Water and extracted With methylene chloride. The organic layer is Washed with dilute hydrochloric acid, dilute sodi um bicarbonate and water until neutral. The dried solution is filtered and evaporated to a residue which is crystallized from aqueous acetone to give 1,4,6-pregnatriene- 14a,17a,21-triol-3,11,20-trione 21-propionate.

EXAMPLE 11 1,4,6-pregnatriene-11[3,14 ]7u,21-tetr0l-3,20-dione and 1,4,6-pregnatriene-85J 1B,] 7a,21-tetr0l,3,20-di0ne The procedure of Example 9 is repeated using as substrate 1,4,6-pregnatriene-115,17a,21-trio1-3,20-dione in the same amounts. The residue is chromatographed as above and washing with 24% methanol in methylene chlorideelutes fractions which on evaporation and crystallization from methylenechloride-hexane give the desired product, 1,4,6-pregnatriene-11/3,14u,17ot,21-tetrol- 3,20-dione. Further Washingwith 46% methanol in methylene chloride elutes crystalline material Which on crystallization from methylene chloride-hexane gives 1,4,6-pregnatriene-8,B,115,17a,21-tetrol-3,20-dione.

EXAMPLE 12 1 ,4,6-pregnatriene-1 1 [3,1 4 11,1 701,21 -tetr0l-3 ,ZO-dione 21 -caproaze Using the procedure of Example 10 with the 14a-hydroxy compound of Example 11 and caproyl chloride, the desired product is obtained. It is crystallizable from methanol and aqueous acetone.

EXAMPLE 13 Qa-fluoro-I ,4,6-pregnatriene-14a,1 70:,21- trial-3,1 1,20-trz'0ne A mixture is prepared of 1% yeast extract concentrate including 90 m1. of an 0.2 M KH PO and 90 ml. of 0.2

um is, sterilizedaby 'auto'claving for 15. minutes at C.

1 and the cooled broth in each :tlask is, inoculated with 1 ml.

of asuspension of (Jurvularia lunata from a 24-hour broth culture; The mixtures are incubated on a shake table for 20 hours at 28 C. After incubation each flask is treated with a solutioncontaining 50 mg. of sterile 9afluoro;1,4,6pregnatriene-1'7a,21-diol-3,11,20:trione in 5 nil. of ethanol. The pH is 6.8-7.2. The cultures are then incubated'andshaken for 48 hours at 28 C. The pH is 7 .07.4. The cultures are then extracted thoroughly with chloroform. The extracts are combined and evaporated to dryness, giving a residue 'of ca. 500-600 mg.

The crude extract is triturated with methanol giving a crystalline solid which: is crystallized from acetone to give 9a-fluoro-1,4,6-pregnatriene l4oc,17oc,2l triol 3,11,20- trione.

EXAMPLE 14 is obtained which. is crystallized from acetone-hexane to give 9a bromo-1,4,6-pregnatriene-11fi,1.4u,17a,21 tetrol- 3 ZO-dione;

EXAMPLE 16 9a-ch loro-Z ,4,6.-prgridtriene-1 40:,1 704,21-

trial-3,1 1,20-tri0ne Using the procedure of Example 13 with 9a-cl1loro- 1,4,6-pregnatriene-17a,2l-diol-3,11,20-trione, a product is I obtained which oncrystallization from aqueous acetone gives 9a-c'hlo'r'o-1,4,6pregnatriene-14a,17a,21 triol-3,11,- 20-trione.

EXAMPLE 17 21 acetates of 14a-hydr0xy compounds Using the procedure of Example 10 with acetic anhydride in place of propionyl chloride, a residue is-obtained, which gives on crystallization from methanol 1,4,6- pregnatriene-14a,17u,21-triol-3,11,20-trione 21-acetate.

The same procedure applied to the l4a-hydroxy' compound of Example 11 gives 'on crystallization from acetone 1,4,6 pregnatriene 11,3,l4ot,l7u,21 tetrol-3,20- dione ZI-acetate.

The same procedure With-the product of Example 13 gives on crystallization from acetone-hexane 9a-flu'or'o- .1,4,6-pregnatriene-14d,17ot,21 triol 3,11,20 trione 21- ac'et'ate.

The same procedure with the product of Example 14 gives on crystallization .from acetone 9a flu0r0'-l',4,6-

pregnatriene-l1B,14a,l7a,21-tetrol-3,20-dione ,21-acetate;

EXAMPLE 1% ],4,6,8 (14) -pregnatetraene-1-1B,1 76:,21-tfi0l-3J0-diofle 1,4,6-pregnatriene-1-1;3,14a,17a,21 tetrol 3,20 dione (0.5 g.) is vdissolvedin 20 ml. of methanol andnitrogen is bubbled through the solution. To this is added a solution of 0.14 g. of potassium bicarbonate in 1 ml. of water and the .mixtureis stirred for eight hours at room temperature. 'The solution is concentratedin vacuo and addition of 'Water. gives crystals which are collected and 11 dried. Recrystallization from acetone-hexane gives 1,4,- 6,8(14)-pregnatetraene-115,17u,21-triol-3,20-dione.

The same product is obtained by the same procedure when 0.5 g. of 1,4,6-pregnatriene-11,6,14a,17a,21-tetrl- 3,20-dione 21-acetate is used as the starting material.

EXAMPLE 19 1 ,4,6,8 (14) -pregnatetraene-1 70:,21-di0l-3,11,20-trione 1,4,6-pregnatriene 14a,17a,21 triol 3,11,20 trione (0.5 g.) is heated at 90-100 C. for /2 hour in 5 ml. of 95% acetic acid. Five m1. of water is added and the mixture is cooled to give crystals which are filtered ed and dried. Recrystallization from acetone gives 1,4,6,8- (14 -pregnatetraene- 1711,21 -diol-3 ,11,20-trione.

When this process is applied to 0.5 g. of 1,4,6-pregnatriene-14a,l7u,21-triol-3,11,20-trione 21-acetate, dilution with water gives a product which is collected, dried, recrystallized from methanol to give 1,4,6,8(14)-pregna tetraene-17a,21-diol-3,11,20-trione 21-acetate.

EXAMPLE 20 9oc-flu0r0-1 ,4,6,8 (14 -pregnatetraene-1 113,1 7:1,21- trial-3,20-di0ne A sample of 0.5 g. of 9a-fluoro-1,4,6-pregnatriene-11p,- 14oz,17ot,2l-llIOl-3,20-di01'l6 21-acetate is treated as in Ex ample 19 leading to a product which, on recrystallization from methanol gives 90: fiuoro 1,4,6,8(14)-pregnatetraene-l1 8,17a,21-triol-3,20-dione ZI-acetate.

A sample of 0.2 g. of this substance is dissolved in 8 ml. of methanol and nitrogen is bubbled through. To the solution is added 0.53 g. of potassium bicarbonate in 0.53 ml. of water and the mixture is maintained under nitrogen for 4 hours. Dilution with water gives a solid which is collected, dried and recrystallized from aqueous acetone to give 9a-fluoro-1,4,6,8(14)-pregnatetraene-11 3,17|x,21- triol-3,20-dione.

EXAMPLE 21 9a-brom0-1 ,4,6,8 (14) -pregnatetraene-1 15,1 70:,21-

trial-3,20-a'ione 21 -acetate A sample of 0.4 g. of 9a-bromo-1,4,6-pregnatriene-11,8,- 14a,17a,2l-tetrol-3,20dione is dissolved in 4 ml. of acetic anhydride While heating to 90-100 on a steam bath. for

1 hour. The solution is cooled, treated with 4 ml. of methanol and poured into 40 ml. of water. The precipitate is filtered off, dried and recrystallized from aqueous methanol to give 9ot-bromo-1,4,6,8(14)-pregnatetraene- 11 19,1 7cz,21-tfiO1-3 ,ZO-dione 21-acetate.

EXAMPLE 22 EXAMPLE 23 9a-chl0ro-1 ,4 ,6,8 1 4 -pregnatetraene-1 711,21 di0l- 3,11,20-tri0ne A sample of 0.2 g. of 9u-chloro-1,4,6-pregnatrienel4a,17a,21-triol-3,11,20-tri0ne is treated as in Example 19. The product obtained on precipitation with water is crystallized from acetone-hexane to give 9a-chloro- 1,4,6,8(14)-pregnatetraene-J113,1Zu,21-tri0l-3,20-dione 21- (5-tert.-butyl)furotzte, 21 -acetate, and 21 -heptan0ate A sample of 0.5 g. of the product of Example 18 is dissolved in 5 ml. of dry pyridine and chilled to 5. To this is added 0.3 g. of 5-tert.-butylfuroyl chloride and the mixture is stirred 15 minutes at 0 and 30 minutes at room temperature. The mixture is poured into 50 ml. of water and the dried precipitate is crystallized from methanol to give the desired ester.

The same procedure using 0.25 g. of acetic anhydride in place of tert.-butylfuroyl chloride gives the corresponding 21-acetate which is recrystallized from methanol.

This application is a continuation of our application, Serial No. 581,265, filed April 30, 1956, which itself is a continuationin-part of our application, Serial No. 559,514, filed on January 17, 1956, now abandoned.

We claim:

1. Compounds of the group consisting of unsaturated pregnanes of the formula wherein X is a member of the group consisting of O and (H, OH), Z represents a configuration of carbon atoms 8, 9 and 14 of the group consisting of W CiJ\/OH (MK/C14 Ca Cs W is a member of the group consisting of H and halogen having an atomic number less than 53, and the 2l-lower alkanoic and 17,21-dilower alkanoic esters thereof.

2. 9ot-Q-l,4,6,8(14)-pregnatetraene 11/3,l7a,2l-triol- 3,20-dione wherein Q represents a halogen having an atomic number less than 53.

3. 9a Q 1,4,6,8(l4) pregnatetraene 1704,21 diol- 3,11,20-trione wherein Q represents a halogen having an atomic number less than 53.

4-. 21-lower alkanoyl esters of tctraene-l1fi,l7a,2l-triol-3,20-dione.

5. 21-1ower alkanoyl esters of l,4,6,8(14) -pregnatetraene-l1,8,17a,21-triol-3,20-dione.

6. 21-lower alkanoyl esters of l,4,6,8(9) -pregnatetraene-17a,2l-diol-3,11,20-trione.

7. 21-lower allranoyl esters of 1,4,6,8(14) -pregnatetraene-17tt,21-diol3,11,20-trione.

8. 2l-lower alkanoyl esters of 9a-Q- l,4,6,8(l4) pregnatetraene-l1fi,17 x,2l-triol 3,20 dione, wherein Q represents a halogen having an atomic number less than 53.

9. 2l-lower alkanoyl esters of 9a-Q-l,4,6,8(14)-pregnatetraene-17a,21-diol-3,l1,20-trione, wherein Q represents a halogen having an atomic number less than 53.

10. 1,4,6,8(9) -pregnatetraene-l7a,2l diol 3,11,20- trione.

11. 1,4,6,8(9)-pregnatetraene-115,17u,2l triol 3,20- dione.

l2. 1,4-,6,8(9) -pregnatetraene-lhll diol 3,11,20- trione 2l-acetate.

l,4,6,8(9) pregna- 

1. COMPOUNDS OF THE GROUP CONSISTING OF UNSATURATED PREGNANES OF THE FORMULA 